Wang Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 124-135). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Table of contents / List of figures / List of tables / List of abbreviations / CHAPTER / Chapter 1. --- Introduction / Chapter 1.1 --- Background of SARS and epidemiology / Chapter 1.2 --- SARS symptoms and infected regions / Chapter 1.3 --- SARS virus / Chapter 1.4 --- Treatment for SARS at present / Chapter 1.5 --- Vaccine development is a more effective way to fight against SARS / Chapter 1.6 --- Vaccine candidates / Chapter 1.6.1 --- Truncated S protein as a vaccine candidate / Chapter 1.6.2 --- Full-length N protein as a vaccine candidate / Chapter 1.7 --- E.coli expression system / Chapter 1.8 --- Baculovirus expression system / Chapter 1.8.1 --- Characteristics of baculovirus / Chapter 1.8.2 --- Infection cycle of baculovirus / Chapter 1.8.3 --- Control of viral gene expression in virus-infected cells / Chapter 1.8.4 --- Merits of baculovirus expression system / Chapter 1.9 --- Aim of study / Chapter 2. --- "Bacterial expression and purification of rS1-1000(E), rS401-1000(E) and rN(E)" / Chapter 2.1 --- Introduction / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Reagents for bacterial culture / Chapter 2.2.2 --- Reagents for agarose gel electrophoresis / Chapter 2.2.3 --- 2'-deoxyribonucleoside 5'-triphosphate (dNTP) mix for polymerase chain reaction (PCR) / Chapter 2.2.4 --- Sonication buffer / Chapter 2.2.5 --- Reagents for immobilized metal affinity chromatography (IMAC) purification / Chapter 2.2.6 --- Reagents for gel filtration chromatography / Chapter 2.2.7 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) / Chapter 2.2.8 --- Reagents for Western blotting / Chapter 2.3 --- Methods / Chapter 2.3.1 --- General techniques in molecular cloning / Chapter 2.3.2 --- "PCR amplification of the S1-400,S401-1000" / Chapter 2.3.3 --- Construction of clone pET-S 1-400 and PET-s401-1000 / Chapter 2.3.4 --- Construction of clone pAC-N / Chapter 2.3.5 --- Expression / Chapter 2.3.6 --- Inclusion bodies preparation / Chapter 2.3.7 --- Inclusion bodies solubilization using urea / Chapter 2.3.8 --- Protein refolding by rapid dilution and dialysis / Chapter 2.3.9 --- Purification of recombinant protein by nickel ion chelating Sepharose fast flow column (IMAC) / Chapter 2.3.10 --- Gel filtration chromatography for further purification / Chapter 2.3.11 --- Bradford assay for the protein concentration analysis / Chapter 2.3.12 --- Protein analysis / Chapter 2.4 --- Results / Chapter 2.4.1 --- SDS-PAGE analysis of the expressed proteins / Chapter 2.4.2 --- Western blot analysis of the bacterial cell lysate / Chapter 2.4.3 --- Protein purification by IMAC / Chapter 2.4.4 --- Purification of rS401-1000(E) by gel filtration / Chapter 2.4.5 --- Determination of production yield of recombinant fusion proteins / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Expression vector selected for rS1-400(E) and rS401-1000(E) expression / Chapter 2.5.2 --- Protein expression in E.coli / Chapter 2.5.3 --- Purification process / Chapter 3. --- Baculovirus expression and purification of rS401-1000(ACN) and rN(BMN) protein / Chapter 3.1 --- Introduction / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Reagents for insect cell culture and virus work / Chapter 3.3 --- Methods / Chapter 3.3.1 --- "PCR amplification of N and cloning of S401-1000, N genes into the transfer vector pVL1393" / Chapter 3.3.2 --- Cloning of S401-1000 into transfer vector pFastBac HT B / Chapter 3.3.3 --- Virus works / Chapter 3.3.4 --- Identification of recombinant BmNPV or AcMNPV / Chapter 3.3.5 --- Manipulation of silkworm / Chapter 3.3.6 --- Mouse immunization for polyclonal antibody against rN(E) protein / Chapter 3.4 --- Results / Chapter 3.4.1 --- Expression of rN(BMN) in baculovirus / Chapter 3.4.2 --- Expression of rS401-1000(BMN) and rS401-1000(ACN) in baculovirus / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- The expression level of rN(BMN) in both in vitro and invivo / Chapter 3.5.2 --- The rS401-1000(ACN) protein expression level in vitro / Chapter 3.5.3 --- Failure in generating rS401-1000(BMN) / Chapter 3.5.4 --- Purification process of rN(BMN) by IMAC / Chapter 4. --- "Characterization of recombinant rS1-400(E), rN(E), rN(BMN), rS401_1000(E) and rS401-1000(ACN)" / Chapter 4.1 --- Introduction / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Reagents for enzyme-linked immunosorbent assay (ELISA) / Chapter 4.2.2 --- Reagents for purification of human IgG / Chapter 4.2.3 --- Source and identity of Immune sera / Chapter 4.3 --- Methods / Chapter 4.3.1 --- ELISA / Chapter 4.3.2 --- Purification process of human IgG / Chapter 4.4 --- Results / Chapter 4.4.1 --- Validation of Immune sera using SARS viral lysate / Chapter 4.4.2 --- Immunoreactivities of rS1-400(E) and rN(E) against pooled patients sera and normal human serum / Chapter 4.4.3 --- Immunoreactivity comparison of rN(E) and rN(BMN) / Chapter 4.4.4 --- Comparison of the immunoreactivities of rS401-1000(E) and rS401-1000(ACN) / Chapter 4.4.5 --- Immunoreactivity of SARS related proteins against Anti-SARS Antibody (Equine) / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Comparison of the immunoreactivities of SARS related proteins expressed in the present study / References
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325167 |
| Date | January 2005 |
| Contributors | Wang, Ying., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xx, 149 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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